High prevalence of human polyomavirus JC VP1 gene sequences in pediatric malignancies.

The oncogenic potential of human polyomavirus JC (JCV), a ubiquitous virus that establishes infection during early childhood in approximately 70% of the human population, is unclear. As a neurotropic virus, JCV has been implicated in pediatric central nervous system tumors and has been suggested to be a pathogenic agent in pediatric acute lymphoblastic leukemia. Recent studies have demonstrated JCV gene sequences in pediatric medulloblastomas and among patients with colorectal cancer. JCV early protein T-antigen (TAg) can form complexes with cellular regulatory proteins and thus may play a role in tumorigenesis. Since JCV is detected in B-lymphocytes, a retrospective analysis of pediatric B-cell and non-B-cell malignancies as well as other HIV-associated pediatric malignancies was conducted for the presence of JCV gene sequences. DNA was extracted from 49 pediatric malignancies, including Hodgkin disease, non-Hodgkin lymphoma, large cell lymphoma and sarcoma. Polymerase chain reaction (PCR) was conducted using JCV specific nested primer sets for the transcriptional control region (TCR), TAg, and viral capsid protein 1 (VP1) genes. Southern blot analysis and DNA sequencing were used to confirm specificity of the amplicons. A 215-bp region of the JCV VP1 gene was amplified from 26 (53%) pediatric tumor tissues. The JCV TCR and two JCV gene regions were amplified from a leiomyosarcoma specimen from an HIV-infected patient. The leiomyosarcoma specimen from the cecum harbored the archetype strain of JCV. Including the leiomyosarcoma specimen, three of five specimens sequenced were typed as JCV genotype 2. The failure to amplify JCV TCR, and TAg gene sequences in the presence of JCV VP1 gene sequence is surprising. Even though JCV TAg gene, which is similar to the SV40 TAg gene, is oncogenic in animal models, the presence of JCV gene sequences in pediatric malignancies does not prove causality. In light of the available data on the presence of JCV in normal and cancerous colon epithelial tissue and our data on amplification of JCV from the cecum of an HIV-infected pediatric patient, further studies are warranted on the role of colon epithelium in the pathogenesis of JCV infection.

JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells.

Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.

Purified JC virus T and T' proteins differentially interact with the retinoblastoma family of tumor suppressor proteins.

The amino termini of polyomavirus T antigens contain LXCXE and J domains, which are necessary for binding and inactivating the retinoblastoma family of tumor suppressors. Both of these motifs are found in the JC virus (JCV) early proteins T'(135), T'(136), and T'(165), leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating function of the JCV large T antigen (TAg). To investigate this hypothesis, the three JCV T' proteins were produced in a baculovirus expression system and purified by immunoaffinity chromatography. To facilitate purification, hybridomas that secrete antibodies recognizing amino-terminal epitopes of JCV early proteins were produced. Potential interactions between the early viral proteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAg and T' proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosphorylated species of the cellular proteins and exhibited the highest binding affinity to p107 and the lowest affinity to pRB. TAg and T'(165) bound more pRB and less p107 than did T'(135) and T'(136); T'(165) also bound less p130 than the other three early proteins. Results of these in vitro interactions were compared to those obtained in vivo using POJ cells, a transformed human glial cell line that expresses JCV early proteins, relatively high levels of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylated p130 is detected in the transformed cells, and most of this protein was found in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins.

Identification of archetype and rearranged forms of BK virus in leukocytes from healthy individuals.

BK virus (BKV) establishes a persistent, asymptomatic infection in more than 80% of the human population, and in immunocompromised individuals BKV causes acute urinary tract infections. Two forms of BKV, archetype and rearranged, have been recovered previously from urine samples. The rearranged form is believed to have emerged from the archetype form by the deletion and duplication of sequences within the transcriptional control region (TCR). We have PCR-amplified unique rearranged forms of the BKV TCR from peripheral blood leukocytes (PBLs) of healthy donors. By employing archetype-specific PCR primers, the archetype BKV TCR was also detected in PBLs. These findings are consistent with the hypotheses that PBLs transport BKV to different sites within the body and play a role in the TCR rearrangement process. BKV sequences were detected in 21 of 38 BKV-seropositive and 1 of 2 BKV-seronegative individuals. Because of recent suggestions that SV40 may circulate in the human population, the donors' sera were also examined for the presence of specific anti-SV40 antibodies. A high antibody titer to SV40 was detected in only 1 of the 40 donor specimens. This study is the first to characterize multiple BKV TCR variants in PBLs from healthy people and to correlate PCR and serological methods used to detect BKV infections.

Identification of JC virus variants in multiple tissues of pediatric and adult PML patients.

The transcriptional control region (TCR) of JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), undergoes rearrangement during replication of the virus in its human host. The mechanism by which viral promoter/enhancer sequences are deleted and duplicated within the TCR of the archetype form of JCV is not understood, but it is hypothesized that the generation of JCV variants with rearranged TCRs contributes to the virus's pathogenic potential. In a recent study of a pediatric PML patient, we detected extensive rearrangement of the JCV TCR in multiple tissues, and the archetype TCR was amplified from sites other than the kidney. These findings differed from those of previous studies that had examined tissues from adult PML patients. Since exposure to JCV usually occurs early in life, it is likely that some pediatric cases of PML arise as the result of a primary infection, whereas adult cases of PML are thought to result from the reactivation of an infection suffered as an immunocompetent child. To investigate further whether rearrangement of the JCV TCR is affected by the host's age and immune status at the time of exposure, a second pediatric patient and two adult PML patients were examined. As in our first study, multiple tissues were found to contain JCV DNA; however, fewer rearranged variants were detected. In one adult patient, related rearranged variants were detected in the brain, while archetype JCV was found in the other tissues. Based on differences in their VP1 sequences, these two forms represented different JCV genotypes, indicating that this patient had suffered a dual infection. The relevance of these findings to the rearrangement process that alters the JCV TCR is discussed.

Rearranged and chimaeric primate polyomavirus genomes.

Recombination between different primate polyomavirus genomes (SV40, JCV, BKV) or within the genome of the same species (e.g. archetype to rearranged type virus) might contribute to the establishment of SV40-like viruses within the human population. Alternatively, it is possible that these polyomaviruses might, upon co-infection of human cells, complement each other's growth through direct virus-virus interactions or by indirect effects on host cell permissiveness. Our laboratory has investigated the activity of JCV-BKV-SV40 chimaeras constructed in vitro, and some of them exhibit altered lytic, host range, and oncogenic behaviour. Our work has also included the PCR analysis of human tissue specimens for the presence of polyomaviral sequences. Archetype and rearranged variants of JC virus have been detected in normal and diseased tissues, but evidence for naturally arising JCV-BKV or JCV-SV40 recombinant genomes has not been obtained.

Identification of SV40 in brain, kidney and urine of healthy and SIV-infected rhesus monkeys.

Recent reports of simian virus 40 (SV40) sequences in human tumors have prompted investigations into the poorly understood association of this polyomavirus with its primate host, the rhesus monkey (Macaca mulatta). In the present study we have used PCR to analyze tissues from 20 monkeys for the presence of SV40. Five of the animals, which were infected with simian immunodeficiency virus (SIV), were found to exhibit SV40-induced lesions and to have SV40 sequences present in their kidney and brain. Lesions associated with SV40 were not observed in 15 SIV monkeys, and SV40 DNA was detected in kidney and urine of only one of these animals. Three regions of SV40 DNA were examined in each tissue: the non-coding transcriptional control region (TCR), the sequences encoding the host range domain (HRD) within the carboxy-terminus of T antigen (TAg), and a portion of the VP1 gene. Each region contained nucleotide alterations compared to the SV40 reference strain 776. In all six animals, the TCR had an archetype structure containing a single 72 bp enhancer element. In addition, the TCR amplified from two animals lacked one of three copies of a GC-rich 21 bp repeat which is part of the promoter in strain 776. Multiple clones of unique HRD sequences were derived from different animals, and in some instances from the same animal. No correlation was found between a particular HRD sequence and its presence in a specific tissue type. Nucleotide changes identified within the VP1 gene indicate that this region, as with the closely-related human polyomavirus JCV, may permit the typing of the virus into individual strains. This study is the first to characterize SV40 sequences present in both healthy and SIV-infected animals and supports the suggestion that strain 776 is not the predominant type of SV40 circulating in its natural host.

Detection of archetype and rearranged variants of JC virus in multiple tissues from a pediatric PML patient.

JC virus (JCV) establishes persistent infections in its human host, and in some immunocompromised individuals, the virus causes the fatal brain disease progressive multifocal leukoencephalopathy (PML). Two forms of the virus, archetype and rearranged, have been isolated, with the latter being derived from the archetype form by deletion and duplication of sequences within the viral transcriptional control region (TCR). We have used the PCR technique to amplify JCV TCR sequences present within multiple tissues of a pediatric PML patient and have cloned and sequenced the PCR products. Archetype JCV was readily detected in kidney tissue; this form of JCV was also identified for the first time in brain and lymph node tissue by employing archetype-specific PCR primers. In addition, several archetype-like variants containing small deletions within their regulatory regions were isolated from cardiac muscle and lung. Different, but related rearranged forms were detected in most of the tissue examined. Each of the rearranged TCRs lacked portions of a 66 base pair (bp) region found within the archetype promoter-enhancer but retained a 23 bp region that is deleted in the prototype (Mad 1) rearranged form of JCV. Although several rearranged forms of JCV were identified in this patient, the TCRs could be assigned to one of two groups based upon the deletion boundaries generated during the adaptation from archetype to rearranged JCV. This study is the first to characterize multiple JCV variants present in different tissues from a patient likely to have succumbed to PML during a primary infection.

Replication activity of JC virus large T antigen phosphorylation and zinc finger domain mutants.

The replication potential of the human polyomavirus JC virus (JCV) relative to that of the related monkey virus SV40 is limited, in part, by differences in the multifunctional T antigen (T Ag). Earlier genetic analyses of the SV40 T protein indicated that specific phosphorylation sites and a zinc finger motif are involved in the regulation of viral replication. The JCV and SV40 T Ags differ with respect to sequences encoding these functional domains, and in the present study mutational analysis of the JCV protein was conducted to assess the role that unconserved residues might play in the restricted lytic behavior of JCV. Amino acids Asn316 and His317 in the zinc finger domain and Thr664 and Glu666 in the carboxy-terminal phosphorylation domain were mutated to either an SV40-like residue or an alanine. Each of the mutant JCV genomes replicated with wild type efficiency suggesting that, unlike the case for SV40 T Ag, these amino acids are not critical to the regulation of viral replication. On the other hand, mutation of amino acid Thr125 within the amino-terminal phosphorylation domain abolished JCV DNA replication and viability. This site is conserved in the SV40 T Ag, and previous results have revealed that phosphorylation of this residue (Thr124) is required for T Ag replication function.

Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions.

The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo.

Sequences within the early and late promoters of archetype JC virus restrict viral DNA replication and infectivity.

Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype's inactivity relative to that of rearranged forms was due to differences in the promoter-enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter-enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s).

Biochemical characterization and localization of JC virus large T antigen phosphorylation domains.

Large T antigen (T Ag), the major regulatory protein produced by the primate polyomaviruses, is a multifunctional phosphoprotein expressed early in the viral life cycle. T Ag performs many functions essential to viral DNA replication, and studies with SV40 T Ag indicate that the regulation of these functions is modulated, in part, by the phosphorylation status of this oncoprotein. In this study, we demonstrate that JC virus (JCV) T Ag obtained from lytically infected and transformed cells is phosphorylated at serine and threonine residues. Analysis of JCV T Ag via two-dimensional tryptic peptide mapping generates 14 phosphopeptides. Additional mapping studies of intact, hybrid, mutant, and truncated forms of JCV T Ag have aided the localization of phosphorylation sites to the N- or C-terminal region of the protein; both serine and threonine residues are modified at each terminus. The data indicate that, unlike the corresponding regulatory phosphorylation site Ser677 in SV40, Thr664 is not phosphorylated in JCV T Ag. The phosphorylation sites utilized for JCV T Ag, and the regulatory role of these sites, are predicted to contribute to the unique biology of this human virus.

Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA.

The genome of the human polyomavirus JC Virus (JCV) encodes two regulatory proteins, large and small T antigen which are expressed early in a lytic infection, and three structural proteins, VP1, VP2, VP3, which are produced late in an infection. A fourth late protein, agnoprotein, may contribute to the assembly of the virion. In this study, we demonstrate the presence of three additional early proteins, T'135, T'136, and T'165, which are expressed in lytically-infected cells; T'135 is also readily detected in JCV transformants. The three species of T' are phosphoproteins generated via an alternative splicing mechanism. This mechanism involves the excision of a second intron from the large T mRNA using a common donor splice site at JCV nucleotide 4274 and three unique acceptor splice sites at nucleotides 2918, 2777 and 2704 for T'135, T'136 and T'165, respectively. The mutant JCV delta T' was created by converting the G at nucleotide 4274 to an A, thereby disrupting the consensus sequence of the shared donor splice site without altering the amino acid sequence of any early JCV protein. Upon transfection of permissive human brain cells, JCV delta T' replicated its DNA 10-fold less efficiently than did wild type JCV. Passage of extracts of the infected cells on to fresh human brain cells revealed that the expression of T antigen was greatly reduced and the presence of T' proteins was undetectable in the mutant versus wild type JCV-infected cells.

Immortalization of human cells by mutant and chimeric primate polyomavirus T-antigen genes.

Human fibroblasts were morphologically transformed with wild type and mutant SV40 T-antigens (T-Ags) and with SV40/JCV and SV40/BKV chimeric T-Ags. The transformants were then assayed for the attainment of immortal cell growth. Several observations relating T-Ag and T-Ag domains to immortalization were made. Approximately 10% of SV40-transformants became immortal. Transformants generated by transfection or infection of cells with C-terminal T-Ag deletion mutants of SV40 did not immortalize. SV40/JCV and SV40/BKV chimeric T-Ags, containing C-terminal sequences from JCV or BKV, immortalized cells more efficiently than did the intact SV40 T-Ag, suggesting that the C-termini of the JCV and BKV T-Ags contain an enhanced immortalization function. However, chimeras in which the N-terminal or proximal-central portions of T-Ag were composed of JCV sequences failed to immortalize but did induce transformation. Constructs in which the JCV T-Ag Rb binding domain was replaced with SV40 sequences transformed human cells, but again the cells failed to immortalize. Transformants and immortalized cell lines produced by some SV40/JCV chimeras, contained p53 which was unbound by T-Ag. This occurred under conditions where p53 from SV40 and SV40/BKV transformants was bound to T-Ag. This may reflect the reduced stability of the SV40/JCV T-Ags.

Cooperative action of cellular proteins YB-1 and Pur alpha with the tumor antigen of the human JC polyomavirus determines their interaction with the viral lytic control element.

Human JC polyomavirus (JCV) is the etiologic agent of the neurodegenerative disease progressive mulifocal leukoencephalopathy. By using JCV as a model, we investigated the role of the viral early protein tumor antigen (TAg) in the binding of two cellular proteins, Pura alpha and YB-1, to JCV regulatory sequences. Results from band-shift assays with purified YB-1, Pur alpha, and TAg indicated that efficient binding of Pur alpha, a strong activator of early gene transcription, to a single-stranded target sequence corresponding to the viral lytic control element, is diminished in the presence of the late gene activator YB-1, which recognizes the opposite strand of the Pur alpha binding site. Of particular interest was the ability of Pur alpha and TAg to enhance binding of YB-1 to DNA molecules without being associated with this complex. Binding studies using a mutant peptide encompassing the N terminus of YB-1 indicated that the C terminus of YB-1 is important for its DNA binding activity. The ability of Pur alpha and TAg to increase binding of YB-1 to DNA is independent of the YB-1 C terminus. Similarly, results from band-shift assays using Pur alpha variants indicated that two distinct regions of this protein contribute either to its ability to bind DNA or to its ability to enhance YB-1 DNA binding activity. Based on the interaction of Pur alpha, YB-1, and TAg, and their binding to DNA, a model is proposed for the role of these proteins in transcription of viral early and late genes during the lytic cycle.

DNA replication of chimeric JC virus-simian virus 40 genomes.

The ubiquitous virus JCV is the etiologic agent of the human brain disease progressive multifocal leukoencephalopathy. Although infection usually occurs early in life and the virus can remain latent in human tissues, including brain, little information is available regarding its replication. It is known that DNA replication of primate polyomaviruses is dependent upon the synthesis of T antigen and the subsequent interactions of this protein with cellular factors and the viral origin of replication. We constructed chimeric genomes between JCV and SV40, two genetically similar viruses with distinct biologies, in which segments of the T antigen coding region and the replication origin were exchanged. Because the engineering of these genomes created a defect in the structural protein VP1, their DNA replicating activities could be compared without the complication of secondary infection of adjacent cells and amplification of the replication signal. The ability of the JCV-SV40 hybrid T antigens to initiate replication from the two viral origins in primate cells was investigated. A region of the JCV T antigen that includes the DNA binding and zinc finger domains was found to be responsible for the failure of JCV T antigen to interact productively with the SV40 origin. In addition, the ability to replicate in monkey cells was limited to constructs expressing T antigens which contained the carboxy-terminal host range domain of SV40.

Converting the JCV T antigen Rb binding domain to that of SV40 does not alter JCV's limited transforming activity but does eliminate viral viability.

Two sets of mutations were introduced into a region of the JC virus (JCV) large tumor (T) antigen involved in binding the retinoblastoma susceptibility gene product (Rb). The first set converted the JCV sequences to those found in the corresponding region of the simian virus 40 (SV40) T antigen. The second set contained sequence changes predicted to abolish Rb binding. Each of these mutations was also inserted into a chimeric T antigen (MSTn) composed of JCV and SV40 sequences at its amino- and carboxy termini, respectively. The JCV T antigen is less efficient than its SV40 counterpart at transforming Rat2 cells and at binding Rb and viral DNA. These activities were altered in the two sets of mutants generated in this study. A JCV T antigen mutant having an SV40-like Rb-binding domain exhibited increased DNA binding activity while, unexpectedly, displaying decreased Rb binding and wild-type transforming behavior. A mutant T antigen that was unable to bind Rb exhibited decreased DNA binding and failed to transform Rat2 cells. Both mutants were defective for DNA replication and did not produce infectious virions. Additional phenotypic changes were observed when each mutation was introduced into the chimeric MSTn T antigen. As the oligomerization state of SV40 T antigen is known to influence several of its activities, including Rb binding, the quaternary structure of the T proteins used in this study was assessed by sucrose gradient sedimentation. The SV40 and chimeric MSTn T antigen sedimented as a mixture of monomers/dimers and higher oligomers, whereas the JCV T antigen sedimented predominantly as monomers/dimers; neither mutation in the T antigen Rb-binding motif affected the sedimentation profiles of the parental T proteins. Restricted biochemical activity of the JCV T protein relative to that of SV40 supports the suggestion that this regulatory protein contributes to the attenuation of the JCV lytic cycle.

Analysis of G418-selected Rat2 cells containing prototype, variant, mutant, and chimeric JC virus and SV40 genomes.

The human polyomavirus JC virus (JCV) is highly tumorigenic in rodents, but transforms cells in culture inefficiently. To explore the basis for JCV's restricted transforming behavior, nonpermissive Rat2 cells were contransfected with pSV2-neo (encodes G418 resistance) and viral DNAs including prototype, variant, and mutant JCV genomes and two JCV-SV40 chimeras. By selecting cells displaying G418 resistance, lines were established that contain viral DNA and exhibit a wide range of transformed phenotypes. The G418-resistant lines were tested for their ability to grow under anchorage-independent conditions, to overgrow a monolayer of untransformed cells, and to form dense colonies on plastic. Expression of the viral T and t proteins and interaction of T protein with the cellular anti-oncoprotein p53 were measured. Also determined was the number of intact viral early coding regions integrated within the cellular DNA. The results of these studies suggested that most of the G418-resistant lines failed to express JCV T protein above a minimum threshold level required for their conversion to a fully transformed phenotype. In anchorage-independent growth assays, higher levels of a 17-kDa T-related peptide in JCV transformants appeared to compensate for decreased T antigen levels. Comparison of the T to p53 ratios in the cell lysates suggested that the quaternary structure of the JCV protein differed from that of its SV40 counterpart in the T-p53 complex. The presence of multiple vs single integrated copies of the viral genome in the cells did not correlate with elevated T antigen expression or an enhanced transformation status.

Expression of viral T-antigen in pathological tissues from transgenic mice carrying JC-SV40 chimeric DNAs.

Immunostaining methods were used to detect viral T-antigen and the cellular protein p53 in pathological tissues obtained from transgenic mice carrying JC-SV40 hybrid viral DNAs. A transgenic mouse carrying the SV40 regulatory region and JC virus (JCV) T-antigen-coding sequences exhibited an SV40-characteristic choroid plexus papilloma that expressed JCV T-antigen and p53. JCV-associated pathology was observed in two other mice in which the JCV regulatory signals directed SV40 T-antigen-induced adrenal neuroblastomas and brain neoplastic cells. However, these mice also exhibited an SV40-characteristic osteosarcoma and abdominal lymphoma that contained SV40 T-antigen and p53-positive cells. Contrasting thymic pathology was observed in the two types of mice where the SV40 regulatory region directed a JCV T-antigen-induced thymoma in one mouse, and the JCV regulatory region directed SV40 T-antigen-induced thymic hypoplasia in two other mice.